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The case of the antibiotic mushroom protein that stood out from the rest

07-06-2022

A collaboration between the Technical University of Denmark, Novozymes A/S and the ESRF leads to the structure of a relevant protein in antibiotic resistance.

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Antibiotic resistance is a major concern among experts due to its increasing prevalence in our health systems. Fifteen years ago, the Danish company Novozymes discovered that Plectasin, a protein from the mushroom Pseudoplectania nigrella, showed antibiotic properties against multiresistant bacteria. And since then, extensive research followed.

It all started when the team was studying the structure of the wildtype Plectasin and three other variants with different amino acids mutated. “We wanted to see how stable they were and how they behave in solution”, explains Pernille Harris, professor at the University of Copenhagen (at the Technical University of Denmark) and corresponding author of the paper. They found that the ones with changed amino acids showed very different stability in solution.

In one of the variants of Plectasin, they noticed a different behaviour: “We found that the protein formed a gel  at neutral and basic pH and wanted to find out how, as it could be important in future applications”, explains Christin Pohl,  now a postdoctoral researcher at Lund University, who was first and co-corresponding author of the paper. When the scientists noticed the gel, they decided to characterise it using AFM and NMR and found out that the protein developed chain-like fibrils. Then they raised the question of whether these were amyloid fibrils, which are present in neurodegenerative diseases like Alzheimer’s disease, or not. The difference between amyloid and non-amyloid fibrils is that in the first type the protein changes its structure to a distinct β-sheet structure, which makes it difficult to reverse this kind of fibril formation, whilst non-amyloid fibrils can be formed by the peptide in its native state. 

But it wasn’t enough. And with samples that didn’t crystallise, they turned to cryo-EM. “I’ve been a long-term user of the ESRF’s Small-Angle X-ray Scattering and Macromolecular crystallography beamlines and I know the quality of the service we get here. So we asked Christoph Müller-Dieckmann for help as we are cryo-EM novices, you could say”, explains Harris. The first contact led to a fruitful collaboration with Müller-Dieckman, Isai Kandiah and the rest of the cryo-EM team at the ESRF. For image analysis Greg Effantin and Guy Schoen from the IBS were involved in this collaboration. “Both teams guided us really well and we managed to solve the structure, which couldn’t have been possible ten years ago”, adds Pohl.

The structure showed that the fibrils are most likely inert but plectasin keeps the antibiotic effect upon release from the fibrils. The scientists believe that they could potentially be used as a slow-release mechanism of the antibiotic property. Sustained release is often facilitated by a release from the surface of a solid particle such as a crystal. The challenge is that the release is proportional to the surface area of the particles which is ever declining as the crystals becomes smaller resulting in a lower and lower release of the active component after dosing. In contrast to most other solid particles release from a fibril can only happen from  the fibril ends and the drug (here plectasin) will be released at a constant velocity facilitating a fully controlled antibiotic concentration throughout the treatment. An additional advantage is the pH tunability of the plectasin fibrils which gives the system an additional controllable parameter for release of the active peptide.

Cryo-EM is a new and very efficient way to get an atomic structure of one-dimensional structures like fibrils and many fibril structures are already published in the literature, which revolutionises the knowledge we have of proteins and peptides in fibrils.

This research was supported by the EU Horizon 2020 research and innovation program (grant agreement no. 675074) in the project PIPPI (Protein-excipient Interactions and Protein-Protein Interactions in formulation).

Reference:

Pohl, C., Effantin, G., Kandiah, E. et al. pH- and concentration-dependent supramolecular assembly of a fungal defensin plectasin variant into helical non-amyloid fibrils. Nat Commun 13, 3162 (2022). https://doi.org/10.1038/s41467-022-30462-w

Text by Montserrat Capellas Espuny

Top image: Isosurface representation of the cryo-EM map along the transverse axis of the fibril. Credits: C. Pohl et al, Nat Commun 13, 3162 (2022). https://doi.org/10.1038/s41467-022-30462-w